The invention provides a composition comprising an aqueous liquid phase, bacterial cells, and an ionic compound dissolved in the liquid phase, characterized in that the ionic compound is selected from the group consisting of 1-butyl-3-methyl-imidazolium-thiocyanate, 1-butyl-3-methyl-imidazolium-2(2-methoxy-ethoxy)ethylsulfate, 1-methyl-1-[4-(3-methyl-3H-imidazol-1-ium)-but-1-yl]-3H-imidazolium-di(toluylsulfate), and 1-butyl-3-methyl-imidazolium-octylsulfate. The compositions of the invention are advantageously used for preparing lysates of biological cells, particularly bacterial cells.
In the biochemical process of purifying an analyte from an intracellular compartment cell lysis is one of the first steps to be performed and of major importance. Concerning the purity and yield of the finally obtained analyte, the way of performing lysis has a significant impact on these parameters. The methods of the state of the art to effect cell lysis are largely based on mechanical and/or enzymatic treatment of the sample material. In addition, various chemical substances are known to the state of the art for the disintegration of cellular structure and the liberation of analytes. Examples therefore are chaotropic agents and detergents. In the case one or more analytes are to be purified from bacterial cells, the skilled person it is faced with a number of problems which are generated by the specific features of these cells.
Bacteria are a large group of unicellular microorganisms. Typically a few micrometers in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals. The bacterial cell is surrounded by a lipid membrane, or cell membrane (also referred to as the cytoplasmic membrane), which encloses the contents of the cell and acts as a barrier to hold nutrients, proteins, nucleic acids and other essential components of the cytoplasm within the cell. As they are prokaryotes, bacteria do not tend to have membrane-bound organelles in their cytoplasm and thus contain few large intracellular structures.
In most but not all bacteria a bacterial cell wall covers the outside of the cell membrane. The cell wall is a tough, flexible or rigid layer that surrounds the cell membrane. Thus, it is located outside the cell membrane and provides the cell with structural support and protection, and also acts as a filtering mechanism. The cell wall is essential to the survival of many bacteria. A major function of the cell wall is to act as a pressure vessel, preventing over-expansion when water enters the cell. Bacterial cell walls are made of peptidoglycan (also known as murein) as the major component. The peptidoglycan is synthesized from polysaccharide chains cross-linked by certain peptides containing D-amino acids. Bacterial cell walls are different from the cell walls of plants and fungi, which are made of cellulose and chitin, respectively. The bacterial cell wall is also distinct from that of Archaea, which do not contain peptidoglycan.
Gram staining is an empirical method of differentiating bacterial species into Gram-positive and Gram-negative, based on the chemical and physical properties of their cell walls. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram variable and Gram indeterminant groups as well.
Gram-positive bacteria possess thick mesh-like cell walls containing many layers of peptidoglycan and teichoic acids. The cell walls of Gram-positive bacteria appear purple upon Gram staining. In contrast, Gram-negative have a thin cell wall consisting of only a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. The cell walls of Gram-negative bacteria stain pink.
In order to purify an analyte from an intracellular compartment of bacterial cells, several methods are known to break up the cells and to release their cytoplasmic components.
Mechanical methods frequently rely on the use of a bead mill break up the material by grinding. At the same time, significant shearing forces are generated. Depending on the analyte to be isolated, shearing is a disadvantage, e.g., when nucleic acids are to be purified.
Liquid-based homogenization is the most widely used cell disruption technique for small volumes. Cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell walls and membranes. Three different types of homogenizers are known in the state of the art. A Dounce homogenizer consists of a round glass pestle that is manually driven into a glass tube. A Potter-Elvehjem homogenizer consists of a manually or mechanically driven pestle shaped to fit a rounded or conical vessel. The number of strokes and the speed at which the strokes are administered influences the effectiveness of Dounce and Potter-Elvehjem homogenization methods. However, owing to largely manual operation, these homogenizers are not suited for high throughput of samples. Also, processing of very small sample volumes is difficult.
A French press consists of a piston that is used to apply high pressure to a sample volume, forcing it through a tiny hole in the press. The French press is often the method of choice for breaking bacterial cells mechanically. However, the device is expensive and also not suited for sample preparation with high throughput.
Sonication is another method of physical disruption commonly used to break open cells. The method uses pulsed, high frequency sound waves to agitate and lyse the bacteria and sometimes even spores. The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy from the probe initiates the formation of microscopic vapor bubbles that form momentarily and implode, causing shock waves to radiate through a sample. To prevent excessive heating, ultrasonic treatment is applied in multiple short bursts to a sample immersed in an ice bath.
Alternatively, the freeze/thaw method can be used to lyse bacterial cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37° C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite lengthy.
In order to make the lysis process more efficient, cells can additionally be incubated with lysozyme (N-acetylmuramide glycanhydrolase), whereby the cell walls are degraded. The enzyme functions by attacking peptidoglycans (found in the cells walls of bacteria, especially Gram-positive bacteria) and hydrolyzing the glycosidic bond that connects N-acetylmuramic acid with the fourth carbon atom of N-acetylglucosamine.
Detergents are other additives used in the processes of lysing a sample containing bacterial cells. However, a detergent frequently produces foam which is not desired in such processes. Also, certain detergents denature proteins. This is a disadvantage in case a protein analyte is purified. The same applies to the use of chaotropic agents as additives.
The methods of the state of the art for lysing bacterial cells have certain disadvantages. It was therefore an object of the invention to provide alternative methods which at least in part overcome these disadvantages. Particularly, it was an object of the invention to provide a method for lysing Gram-positive and/or Gram-negative bacterial cells. It was a further object of the invention to provide a method for lysing bacterial cells in a small volume.
In case the sample material not only comprises bacterial cells but also other cell types, e.g., yeast cells, it is often desired to use the methods for lysing cells which is selective for the target cells. Thereby, the amount of undesired byproducts released from other cells but the target cells can favorably be reduced. A further object of the invention therefore was to provide a method for cell lysis which is specific for bacterial cells but not for other microbial cells such as fungal or yeast cells.